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Abstract:

Dual mode genetics testing systems are devised about a single element
testing platform. A microfluidic network and system of interconnected
receiving cells and reaction vessels supports at the same time genotyping
and copy number analysis where the platform may be subject to a common
thermal cycle schedule to cause the proper reactions (DNA replication)
necessary in both test types. Further, the microfluidic platform which
includes reaction vessels for genotyping which are spatially removed from
reaction vessels for copy number analysis, is coupled to optical scanner
and detection systems specifically arranged to apply test specific
detection routines on each of these distinct regions or portions of the
dual mode test platform.

Claims:

1. Dual mode genetic testing platforms comprising: a microfluidic
platform; a polymerase chain reaction thermal cycler; and a multi-channel
optical detector, said microfluidic platform is comprised of two modules
including: a copy number analysis module; and a genotyping module, said
copy number analysis module is spatially removed and separate from said
genotyping module and each being optically coupled to a separate channel
of the multi-channel optical detector, said copy number analysis module
and said genotyping module are each comprised of a plurality of reaction
vessels, and said copy number analysis module and said genotyping module
are each thermally coupled to said thermal cycler.

2. Dual mode genetic testing platforms of claim 1, said multi-channel
optical detector having a CCD device with a first channel coupled to
reaction vessels of said copy number analysis module and a second channel
coupled to reaction vessels said genotyping module, the reaction vessels
of the genotyping module are spatially removed from reaction vessels of
the copy number analysis module whereby separate optical signals may be
captured with respect to each module.

3. Dual mode genetic testing platforms of claim 2, said first channel
coupled to said genotyping module being further comprised of driver
electronics operable for capturing an optical signal at a discrete moment
in time; and a second channel coupled to said copy number analysis module
being further comprised of driver electronics operable for capturing a
plurality of discrete optical signals over the course of an extended
period of time

4. Dual mode genetic testing platforms of claim 3, said first channel is
further arranged with driver electronics operable for capturing optical
signals of a plurality of wavelengths.

5. Dual mode genetic testing platforms of claim 4, each wavelength
corresponds to a different version of a single nucleotide polymorphism.

6. Dual mode genetic testing platforms of claim 3, said second channel is
further arranged with driver electronics which enable the optical
detector to detect return optical signal of intensities which range over
at least two orders of magnitude.

8. Dual mode genetic testing platforms of claim 7, said. TaqMan chemistry
is provided in two compositions, one TaqMan composition for use with the
genotyping module and another TaqMan composition for use with the copy
number analysis module.

9. Dual mode genetic testing platforms of claim 8, said TaqMan chemistry
composition associated with the copy number analysis module is further
comprised of a contrast agent.

10. Dual mode genetic testing platforms of claim 6, said copy number
analysis module is further comprised of a known reference allele arrange
to react with TaqMan chemistry to produce an optical signal by which a
comparison may be made with respect to alleles under test.

11. Dual mode genetic testing platforms of claim 7, said TaqMan chemistry
includes genetic probes arranged with a plurality of color florochromes
optical reporters--one said optical reporter each for each SNP version.

12. Dual mode genetic testing platforms of claim 1, further comprising a
DNA sample whereby at least one receiving cell of the genotyping module
and at least one receiving cell of the copy number analysis module each
receive a portion of the identical DNA sample.

13. Dual mode genetic testing methods comprising the steps: i) providing
a plurality of receiving cells in two distinct test modules with TaqMan
reagent chemistry, said two distinct test modules including a genotyping
module and a copy number analysis module; ii) providing at least one DNA
sample in a plurality of receiving cells of two distinct test modules
including a genotyping module and a copy number analysis module; iii)
mixing provided TaqMan reagents with at least one sample DNA; iv)
executing a repeated cycle to induce replication of DNA and multiply
copies whereby the cycle includes: a) heating genetic matter in a
reaction vessel to cause denaturing of a DNA sample double helix; b)
cooling genetic matter in the reaction vessel to cause annealing and
replication of DNA sample; c) illuminating the copy number analysis
module with high energy light to stimulate fluorescence in the reaction
cells thereof; d) capturing an optical return signal from the copy number
analysis module; and e) determining whether the last executed cycle is a
final cycle of a prescribed thermal cycle schedule; v) illuminating the
genotyping module with high energy light to stimulate fluorescence in the
reaction cells thereof; and vi) capturing a spatially distributed optical
signal from the genotyping module.

14. Dual mode genetic testing methods of claim 13, said "providing TaqMan
reagent chemistry" step is further characterized as loading receiving
cells of a copy number analysis module with TaqMan chemistry; and loading
receiving cells of a genotyping module with another TaqMan chemistry.

15. Dual mode genetic testing methods of claim 14, said "loading
receiving cells of a copy number analysis module" is further
characterized as providing a TaqMan composition with an additive to
enhance contrast.

16. Dual mode genetic testing methods of claim 13, said "providing at
least one DNA sample" is further characterized as providing a reference
oligo in at least one receiving cell of the copy number analysis module.

17. Dual mode genetic testing methods of claim 13, said "capturing an
optical return signal from the copy number analysis module" is further
characterized as capturing a plurality of signals over the course of a
thermal cycle schedule to form a plot of return signal intensity v.
thermal cycles applied from which a copy number determination may be
based.

18. Dual mode genetic testing methods of claim 13, said "capturing an
optical return signal from the genotype module" is further characterized
as capturing and thresholding an optical signal at the end of a thermal
cycle schedule from which a genotype determination may be based.

19. Dual mode genetic testing methods of claim 17, said capturing an
optical signal from the genotyping module is characterized as capturing a
bi-color signal whereby a determination as to heterozygote, homozygote
major and minor may be made.

20. Dual mode genetic testing methods of claim 13, said "providing at
least one DNA sample" step is further characterized as placing DNA from a
single organism in at least one receiving cell of the genotyping module
and at least one receiving cell of the copy number analysis module.

Description:

BACKGROUND OF THE INVENTION

Field

[0001] The following invention disclosure is generally concerned with
genetic testing and specifically concerned with genetics testing
simultaneously in two modes including a sequencing mode and a copy number
analysis mode.

[0002] Significant scientific advances in genetics processing technologies
and have been aggressively developed by talented scientists in this
modern era of genetic science advance. At the core of some of these
technologies lie certain well-known and common processes including qPCR,
`TaqMan`, among others. A push for efficiency and low cost continues to
drive further improvement and each day new systems arrive which also
improve our genetics testing capabilities.

[0003] In one special scenario of genetics testing strategy, it is
important to perform both genotyping and copy number analysis genetic
tests. This may be particularly the case with respect to a single genome
because these two types of genetics tests rely on states of chemical
reactions having somewhat different physical conditions and attributes,
they were heretofore performed separately. Separate platforms were run in
separate processes where distinct measurements were made for each test.
But running separate tests for genotyping and copy number analysis is
expensive in materials; consumes highly specialized equipment operation
time; and requires significant skilled labor. It is thus highly desirable
to perform both genotyping and copy number analysis in an single process
run. This is especially the case when both genotyping and copy number
analysis are to both be done on a single person's DNA or genome.

[0004] One most important platform for genetics testing is sometimes known
as a "microfluidic genetics testing" system. Genetic samples and
carefully prepared test chemistry (TaqMan) may be combined via a network
of fluid channels to cause reactions between matter from both. Many forms
of these arrays are now readily found in genetics testing laboratories
everywhere.

SUMMARY OF THE INVENTION

[0005] Comes now, Tanya Moreno, Cindy Wang and David Becker with
inventions of genetic testing systems including devices and methods for
genetic sequencing and copy number determinations. It is a primary
function of these systems to simultaneously and on a single platform
provide for genetic measurements relating to both sequencing and copy
number. It is a contrast to prior art methods and devices that systems
first presented here do not require separate machine runs and independent
processing. Rather, a single multiplex operation permits dual measurement
types in a single well configured platform. Specifically, arrays of
microfluidic reaction vessels are coupled to distinct optical detection
means--each of two optical detection means directed to detection schemes
for either sequencing or copy number determination.

[0006] A single microfluidic platform is arranged and devised along with
its cooperating components and supporting apparatus, to perform both
genotyping and copy number analysis in a single process run. An array of
microfluidic cells and coupled fluid circuits forms a dual-mode genetics
testing platform arranged to perform simultaneously genotyping and copy
number analysis on a common genetics test sample set.

[0007] The microfluidic array is adapted to receive therein genetic matter
from one or more individual test subjects of interest. In another portion
of this dual-mode testing platform, chemistry or testing reagents may be
received therein appropriately arranged receiving cells. So received,
test chemistry is mixed with DNA matter after being brought together via
a network of fluid channels to mix reaction vessels. Process steps
including thermal cycles are performed while reagent chemistry and
genetics matter under test are in a common cell to produce a reaction
indicative of certain genetic states or conditions.

[0008] In one illustrative example version, in the portion of the test
platform arranged-to receive test chemistry, two distinct regions may be
defined--each of these regions may include a plurality of receiving
cavities into which reagents may be introduced. However, one first region
associated with genotyping may receive a different chemical preparation
than a second region associated with copy number analysis. The test
chemistry for copy number analysis is distinct from and may sometimes be
unsuitable for use in genotyping tests--and vice-a-versa. Accordingly,
the portion of the microfluidic testing platform arranged to receive test
chemistry is sometimes provided with two discrete and separate regions.
One region each for each type of chemistry associated with genotyping and
copy number analysis.

[0009] Dual-mode microfluidic test platforms first taught here are
additionally arranged whereby they may be simultaneously coupled to a
thermocycler and further coupled to optical detector systems. A
thermocycler applies heat and cooling cycles in a highly regulated manner
to the reaction vessels of the microfluidic platform to affect prescribed
reactions between test DNA and reaction chemistry or chemistries. To
bring about a high performance genotyping test, a particular
heating/cooling schedule is necessary. Similarly, to bring about a copy
number analysis a certain/cooling schedule is required. While
heat/cooling cycles are sometimes quite similar and may in fact be
identical under specific conditions, one important distinction with
regard to the reaction vessels associated with copy number analysis, is
that they must be optically interrogated between each thermal cycle (or
set of thermal cycles) to properly measure the copy number or `real-time
PCR` signal. In genotyping, little or no information may be gained by
measuring the reaction extent prior to completion of the thermal cycle
schedule--by contrast, it is the end result after the entire set of
thermal cycles are applied that is important to the genotype test. Yet it
is impossible to attain copy number information at the end of the thermal
cycle schedule without data taken throughout and during the thermal
process.

[0010] Accordingly, special optical detector array systems are coupled to
the reaction vessels by spatial division and spatial multiplexing. Such
optical detector arrays include pluralities of optical detection elements
in two distinct groups. A first group of optical detection elements is
provided with amplification electronics which may drive the detector
elements in accordance with the heat/cooling cycles to make a plurality
of optical measurements throughout the course of thermocycler
application. That is, this portion of the optical detection system is
coupled to the thermal cycler. This first group of optical detection
elements is associated with copy number analysis may be sometimes
referred in the arts as `qPCR` or `real-time PCR`. These optical
detection elements are coupled to corresponding reaction vessels which
received the copy number reaction chemistry. Reaction vessels having
therein the chemistry appropriate for genotyping are spatially separate
from the copy number analysis reaction vessels and are thus physically
coupled to a different optical detection system. An optical detection
system suitable for genotyping measurement includes an amplifier and
drive electronics which operate to make a single measurement at the end
of the thermal cycle schedule. In addition, this optical detection system
may also support advanced chromatic filtering whereby distinct colors of
optical return signals may be addressed separately. In some genotyping
systems, a single SNP may have either of a (major allele or minor allele)
versions. In these cases, color is sometimes used to distinguish between
these. There is no similar use of chromatic variation in the copy number
portion of the optical detection and that optical detection system
coupled to the copy number reaction vessels may be `colorblind`.

[0011] Accordingly, systems first presented herein this disclosure are
dual-mode genetics testing platforms of two portions, a first arranged to
execute genotyping tests and a second suitably arranged to execute copy
number testing. These both exist together in a single microfluidic
platform and both types of tests may be executed together during a
process thermal cycling and optical integration amenable to both distinct
tests.

[0012] Such devices and systems are therefore well-positioned for cost
savings and genetics testing improvements. Because a single platform is
processed once with a group of DNA samples to produce both genotype and
copy number data for each person under test on a single process run
equipment, consumables and labor are significantly conserved.

[0013] The invention thus stands in contrast to methods and devices known
previously. The invention includes a single apparatus and platform for
carrying out these two important types of genetic tests and the tests may
be executed simultaneously. Systems known today all belong to the body
art which requires two separate processes including one process on one
distinct platform for genetic sequencing and another process on another
platform for copy number variation measurement. Heretofore, these tests
are not made simultaneously on a single platform.

OBJECTIVES OF THE INVENTION

[0014] It is a primary object of the invention to provide new genetic
testing systems.

[0015] It is an object of the invention to provide dual mode genetic
testing systems based upon microfluidic reaction platforms.

[0016] It is a further object to provide microfluidic genetic testing
platforms which operate simultaneously in a genotyping mode and a qPCR
mode.

[0017] It is an object of the invention to provide dual mode microfluidic
test platforms for genetics comprehensive genetics testing.

[0018] A better understanding can be had with reference to detailed
description of preferred embodiments and with reference to appended
drawings. Embodiments presented are particular ways to realize the
invention and are not inclusive of all ways possible. Therefore, there
may exist embodiments that do not deviate from the spirit and scope of
this disclosure as set forth by appended claims, but do not appear here
as specific examples. It will be appreciated that a great plurality of
alternative versions are possible.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0019] These and other features, aspects, and advantages of the present
inventions will become better understood with regard to the following
description, appended claims and drawings where:

[0020]FIG. 1 is a prior art diagram illustrating a popular genetic
testing reaction--TaqMan;

[0021]FIG. 2 is a chart diagram showing a return optical signal taken
over the course of a TaqMan reaction process; and

[0022]FIG. 3 is an illustrative first example of a test platform
including an array of reaction vessels;

[0023]FIG. 4 is a line diagram representation of a dual mode genetics
testing platform showing examples of receiving cells coupled to reaction
cells;

[0024]FIG. 5 illustrates dual optical detection systems spatially
multiplexed to various portions of a dual mode platform of these systems
shown in a cross section view;

[0025]FIG. 6 illustrates example spatial distributions which might be
used in various alternative versions these systems; and

[0026]FIG. 7 is a block diagram setting forth major elements of methods
of these systems including relationships therebetween same elements.

PREFERRED EMBODIMENTS OF THE INVENTION

[0027] As an introduction to one important form of genetic testing, the
reader will appreciate the brief review of TaqMan mechanics
herefollowing. TaqMan probes 1 bind to a long strand of DNA 2. These
probes include a complementary nucleotide combination with respect to the
test DNA to which is binds. The probe additionally includes an optical
marker or `reporter` 3 and further an optical `quencher` 4.

[0028] After test DNA is subjected to high temperature, it is denatured
into two separate strands, the TaqMan probes can anneal themselves
thereto at sites with matching oligos. As the chemistry cools further, a
primer 5 anneals to the template DNA strand to form a complimentary
strand. Taq polymerase 6 adds further nucleotides to the strand until it
reaches the probe and eventually removes the probe from the template DNA
and in the process separating 7 the reporter from the quencher. When
illuminated, a free reporter will re-emit optical energy which can be
detected by an optical sensor or scanner. Further replication of the PCR
product is not interrupted by the probe. The newly formed double strand 8
is ready to be denatured again and the entire process repeated in further
PCR cycles.

[0029]FIG. 2 illustrates a signal which is characteristic of a copy
number analysis. In copy number testing, a DNA test sample is inserted
into a receiving cell or a plurality of receiving cells of the testing
platform designated for that purpose. A receiving cell arranged to
receive the appropriate TaqMan reagent chemistry, a cell cooperatively
coupled to the receiving cell in which the test DNA sample was received
whereby matter received at these two cells are combined in a common
reaction vessel having been previously mapped to a specific known
location of the array platform whereby optical access is afforded. So
combined, the chemistry is subjected to repeated heating and cooling
cycles--or thermal cycles as illustrated in the drawing figure on the
horizontal axis. As thermal cycles are processed, the DNA is replicated
repeatedly and copies 22 (vertical axis of the chart) of the DNA are
formed. In the case where specific conditions are met i.e. the TaqMan
probe matches the test DNA and binds thereto, copies are produced and
optical markers are cleaved away with every thermal cycle applied.
Conversely, where the probe sequences are not found in the sample DNA,
the replication process does not produce appreciable amounts of free
optical reporters.

[0030] If the reaction vessels associated with copy number variation
analysis are appropriately illuminated after thermal each cycle, a return
optical signal from the free reporters proportional to the number of
copies can be detected at optical detector elements arranged to detect
same; i.e. optical detectors having a high intensity dynamic range. In
some test systems, a prescribed number of copies e.g. 100,000 copies 23
may correspond to a threshold signal level 24. If the optical signal 25
returned from copy number analysis reaction vessels reaches the threshold
level after a specific number of cycles 26, it is safe to draw a
conclusion regarding the copy number for the test sample. This is due in
part to a known reference cell run in parallel where the copy number is
known. The relative intensities permits one to conclude copy number
information about the test sequence. While this copy number analysis and
technique may be well known in the art, and therefore it is not the
purpose of this disclosure to detail qPCR, what is not known is using a
microfluidic platform where various portions of the platform are
simultaneously coupled to separate detection systems and technique. That
is, where portions of the platform support copy number analysis and other
portions of the platform are arranged to support genotyping for the same
process run.

[0031] This principle of a dual-mode microfluidic platform 31 arranged to
support at the same time both genotype testing and copy number analysis
testing is better understood in view of the drawing FIGS. 3 and 4. With
reference to FIG. 3, a diagram which details a microfluidic network of
coupled cells, receiving cells 32 are arranged to receive therein DNA
samples to be tested. Samples of DNA from donors of interest may be
inserted into these cells which operate as an input port of the
microfluidic system. DNA which has been subject to preprocessing might
include steps to stabilize, isolate or purify DNA samples. In addition,
the DNA samples may be supported in a chemical medium amenable to DNA
replication processes and conditions. Receiving cells 32 may be arranged
to receive DNA from a single subject i.e. a single person, or arranged to
receive DNA from, several persons. However, each cell typically supports
receipt of DNA from a single organism (person).

[0032] Each of the receiving cells 32 (25 cells in the illustrative
diagram) is coupled by way of tiny fluid channels to an array of reaction
vessels in which chemistry from a plurality of sources (one DNA sample
and one reaction reagent) may be mixed together and further in which
chemical reactions may, be effected. Further, these reaction vessels are
arranged whereby an optical probe and/or optical illumination beams may
be received such that illumination light falls incident upon chemistry
contained in the reaction vessel. Further, these reaction vessels are
carefully coupled to optical detectors which operate to detect light
radiated from the reaction chemistry in response to stimulation by
illumination beams.

[0033] Receiving cells 33 are arranged to receive therein reagents used to
support DNA replication reactions and DNA probes. In some preferred
versions, there are at least two types of important modules arranged to
receive these test reagents. Two distinct compositions of reagents, each
composition supporting either of two types of processes. These include
reagents which support genotype genetics testing methodology and reagents
which support copy number analysis genetics testing.

[0034] These reagents are not mixed together in any single receiving cell,
but rather are physically isolated--a reagents composition suitable for
genotyping being put into receiving cells of the genotyping module and
some another distinct composition of reagents being put into receiving
cells of the copy number analysis module. In this way, we can assure
spatial distinction between the two types of reactions being
simultaneously carried out on the dual-mode platform.

[0035] Because relative intensity of return signals from a copy number
module is critically important for accurate copy number determinations,
it is useful to include in reagent chemistry a contrast enhancing
solution. It is not necessary to include a contrast agent in the TaqMan
used with the genotyping module because optical signals read there are
thresholded and there is no range of intensities to be compared. A
contrast agent does not improve the optical return signal in the
genotyping channel of these optical detectors. Accordingly, only the
reagents used in the copy number analysis module contain such contrast
agent.

[0036] At least one reaction vessel of the copy number analysis module is
reserved for a reference probe. When appropriate reagents are added to
the copy number analysis module, these include genetic probes and a
reference DNA strand. That is, one of the reaction vessels necessarily
includes a genetic probe and reference oligo which will produce a
replication reaction at a known rate. From this reference, all other
optical signals of the copy number analysis module may be compared.
Accordingly, reagents provided to the copy number analysis module also
include this reference scheme. As the optical signals of the genotyping
module are not compared in this way, compositions of reagents provided to
the genotyping module do not include similar reference materials.

[0037] However, genotyping modules do sometimes require unique reagent
chemistries. In some important versions, a genotyping reaction will
include a plurality of distinct optical reporters. These optical
reporters may be distinguished by their color. Each reporter may respond
to the reaction process differently depending upon the precise nature of
the SNP under test. In this way, different colors may be used to indicate
the presence of various zygote forms. Reagent compositions used with the
copy number analysis module does not benefit from the use of
multi-colored reporters. As such, reagent compositions for each of the
two modules may be distinct and these must be properly mapped in
agreement with the spatial distribution schemes of these dual mode
systems.

[0038] Receiving cells which support receipt therein of various reagent
chemistry are coupled by way of microfluidic channels 34 to the array of
reaction vessels. In some versions, a single receiving cell is coupled to
each reaction vessel of an entire row of reaction vessels.

[0039] Similarly, a receiving cell 36 in receipt of DNA test matter may be
coupled to each reaction vessel of an entire row 37 of reaction vessels.
In this way, we can assure that a single DNA sample can be subjected to a
large plurality of different reagent chemistries i.e. 25 in this example
of FIG. 3. Reaction vessel 38 thus receives by way of the microfluidic
channels the DNA sample from receiving cell 36 and reagent chemistry from
receiving cell 39.

[0040] For a complete understanding of these systems, it is important to
appreciate the nature of spatially distinguishing reaction vessels
associated with the two types of genetics testing. A reaction vessel
associated with either test type is chemically coupled by way of fluid
channels to appropriate reagents and is further optically coupled to a
discrete detection system most suitable for the particular test type.
These dual-mode platforms must support simultaneous testing of two types
via spatially distributed elements of an array of reaction vessels which
together form a unitary platform. However, it is a necessary requirement
that the appropriate chemistry and appropriate detection systems--for
each genetics test type be properly mapped to the various reaction
vessels. That is, a reaction vessel associated with genotype testing is
coupled to the appropriate reaction chemistry and appropriate detection
system by way of its unique mapped location in the array. Reaction
vessels associated with copy number analysis similarly are coupled to
appropriate reaction chemistry and an optical detection system suitable
for copy number type measurements. Both types of reaction vessels,
despite their spatial distribution and physical separation are thermally
coupled to the same thermocycler system. It is not necessary in the two
types of tests to apply different thermocycler schedules and a single
thermal cycle schedule can be used to advance appropriate DNA replication
in both genotype tests and copy number analysis tests. For this reason,
these microfluidic platforms may be coupled at their underside to a
single thermocycler which delivers identical heat/cooling cycles to both
types of reaction vessels; i.e. there is no need for spatial distribution
of heat application.

[0041]FIG. 4 presents in an example constructed for illustration purposes
and ease of understanding, twenty-five receiving cells 41 are suitable
for receiving therein DNA samples of either a single test subject or
plurality of test subjects. Ten reagent receiving cells 42 distributed in
a prescribed region 43 comprise receiving cells which support copy number
analysis type genetics testing. A region 44 of fifteen receiving cells
forms a microfluidic platform input port associated with genotype
testing. Chemistry appropriate for genotype testing is received at cells
in this region. Such receiving cells are coupled by fluid channels 45
whereby reagents received therein are conveyed to reaction vessels of the
region 46 shown which form an array of 375 reaction vessels which support
genotyping. An array of reaction vessels forms the region 47, these cells
which are spatially removed and distinct from the genotype reaction
vessels, is comprised of 250 reaction vessels dedicated for copy number
analysis or real-time PCR testing. These vessels receive copy number
reagents by way of micro-channels coupled to the receiving cells of
region 43. In this way, reagents specific to the type of genetics testing
are spatially mapped to particular cells of the reaction portion of the
platform. In a final critical arrangement, reaction vessels elements 46
is optically coupled to a detection system appropriate for genotype
testing and reaction vessels array 47 is optically coupled to a detection
system most appropriate for copy number analysis. It will be understood
that despite the appearance of the diagram which does not show the
micro-channels for all receiving cells, such micro-channels nevertheless
do couple receiving cells to reaction vessels.

[0042]FIG. 5 is provided to disclose details of special optical systems
of these dual-mode microfluidic testing platforms. Of course, the
previous diagrams clearly show how reagents specific to the two types of
genetics testing are coupled to the reaction vessels in a manner whereby
the reagents are easily mapped to the reaction vessels array, those
diagrams do not suggest how spatial separation cooperates with the
distinct detector strategies associated with genotype testing versus
real-time PCR (copy number analysis). As described previously herein, a
detector suitable for copy number analysis requires an electronics driver
system coupled to the thermocycler which makes a plurality of intensity
measurements--one each for each thermal cycle in preferred systems.
Conversely, driver electronics for genotype test detection only need a
single measurement at the end of a plurality of applied thermal cycles.
However, in some genotyping schemes, a plurality of wavelengths may be
necessarily used and a genotype detector must operate in view of
chromatic distinctions which may sometimes be present. Optical detectors
coupled to copy number analysis regions of the array may be color blind.

[0043] Accordingly, these distinct detector characteristics must be
respected in view of the spatial separation of reaction vessels dedicated
to each type of genetics test. To effect this, an example imaging system
is illustrated in FIG. 5. Reaction vessels 51 associated with genotype
testing are imaged at image plane portion 52 by lens 53 which maintain
spatial integrity between object and image planes. Likewise, reaction
vessels 54 associated with copy number testing are imaged at image plane
portion 55. This is better understood by the ray trace 56 diagram well
known in the optical arts. The image plane may share space with optical
detectors such as a silicon photodiodes or the more sophisticated CCD
type pixel imagers. For geometric reference and comparison with earlier
presented figures, DNA sample receiving cells 57 and reagent receiving
cells 58 are shown aside of the reaction vessels array (51 and 54). Two
distinct electrical drivers D1 and D2 are coupled to illumination systems
59 and distinct detector plane image elements by way of amplifier 510 and
amplifier 511. Driver D1 is arranged to illuminate the object plane for
each thermal cycle applied by the thermocycler 512 or more precisely, a
polymerase chain reaction PCR thermal cycler. In addition, the driver D1
is arranged to measure the optical intensity at each image element in the
image plane that is associated by spatial relationship with a reaction
vessel which supports copy number genetics testing. Accordingly, each
optical detector of this portion of the detection system has a high
dynamic range.

[0044] Similarly, driver D2 is arranged to illuminate, reaction vessels
having therein genotype test chemistry. Although a plurality of distinct
chromatic measurements may be made, genotype detection only requires
intensity measurements be made after all required thermal cycles have
been executed. Thus D2 is arranged to illuminate the object plane and
read signals from a CCD in a single measurement operation which occurs
after the thermal cycle schedule is completed. In this way, a single
physical platform is multiplexed whereby spatially distinct regions
permit optical coupling to two different detection systems arranged to
make specific measurements, further whereby mechanical arrangement of
microfluidic channels permit receipt of two types of test chemistry for
reagent multiplexing, and further whereby the platform may be thermally
coupled to a single thermocycler system where required thermal cycling
for both types of genetics test may be applied simultaneously.

[0045] It should be appreciated that the spatial arrangements illustrated
in FIG. 3 and FIG. 4 are not sacred. It is entirely possible and
anticipated that other arrangements including random distributions are
possible. FIGS. 6A to 6D is provided to illustrate. FIG. 6A shows a
uniform 25 x 25 distribution of reaction vessels of a microfluidic array
genetics test platform. Stippling is provided to indicate those cells
dedicated to copy number analysis and those allocated to genotype
testing. Vertical stippling in 250 cells of the left side are those copy
number reaction vessels, while 375 cells on the right half of the
platform comprise reaction vessels of genotype configuration in agreement
with earlier presented figures. However FIG. 7B shows an important
alternative. Cells randomly distributed about the array have vertical
stippling to indicate their type as a copy number test cells. Similarly,
spatially separate a random distribution of cells having horizontal
stippling indicate those cells provided for genotype testing. When drawn
separately, FIG. 6C shows genotyping reaction vessels in FIG. 6D shows
copy number analysis cells. Because a detection system comprised of
discrete picture elements is easily aligned with the test platform
described as such having a plurality of spatially distinct cells, it only
requires a careful mapping in software to drive the detector in an
appropriate fashion for both types of testing. Nevertheless, optical
signals are read from the copy number test cells throughout the thermal
cycles--either between each cycle or between each set of several cycles
to effect a plot as shown in FIG. 2 which is necessary for copy number
analysis.

[0046] While apparatus of these systems were fully and thoroughly
presented in the description previous, methods of these systems follow
closely and may be further characterized as follows. In a first step,
chemistry appropriate for genotype measurements is prepared 71. This may
include chemistry sometimes known as TaqMan. These TaqMan probes include
optically activated markers attached to genetic fragments where the
sequence of the genetic fragment is a combination of particular interest.
In some versions, more than one color of optical marker may be used and
sometimes these are used in to distinguish between major and minor
alleles of the same SNP.

[0047] Similarly, reagents are prepared 72 in support of a copy number
genetics test. These may also be arranged as TaqMan reagents, however
color is unimportant. Rather, in copy number analysis schemes a reference
sequence of known copy number is included to form an intensity comparison
reference. A sequence under test may present a higher density than the
reference where the copy number is higher and lower intensity where the
copy number is lower. Accordingly, reagents appropriate for copy number
testing include this baseline reference.

[0048] TaqMan probes for genotyping tests are distinct from those relating
to copy number. When performing genotyping tests, certain SNPs are far
more interesting than others. The particular nucleotide combinations
which define these SNPs of high interest will be embodied as the TaqMan
probes of a genotype reagent. However, SNPs which are related to copy
number studies are not always the most important SNPs of interest in
genotyping. Therefore, TaqMan probes useful in copy number analysis may
have different nucleotide sequences that those used for genotyping.

[0049] After these two reagent types are prepared, they are distributed 73
to various of the reagent receiving cells in accordance with a prescribed
mapping known to the detection system. In a following step, DNA test
matter is inserted 74 into receiving cells. Reagents and test DNA
together are conveyed from their respective receiving cells via
microchannels to reaction vessels of a reaction vessel array and mixed 75
in another step of these methods. After reagents and test DNA are
together in respective reaction vessels, a thermocycler applies 76
heat/cooling cycles simultaneously to both types of reaction vessels.

[0050] After one cycle, or a prescribed set of cycles are applied,
intensity measurements are made at the copy number reaction vessels. A
conditional step 77 is executed to determine whether the thermal cycle
schedule is completed. If not, a qPCR or real-time PCR measurement 78 is
made. Additional thermal cycles are applied and the same conditional is
repeated. After all cycles have been applied to reaction vessels and the
thermal cycle schedule is complete, a final intensity measurement 79 is
made at each genotyping reaction vessel.

[0051] In this way, a single platform may be used to simultaneously
execute both genotyping and copy number analysis in a single process run.
This is particularly important when DNA from a single person is to be
subject to both tests. Where microfluidic systems are configured for
either test but not the other, then two separate process runs must be
executed and this consumes excess resources. Accordingly testing in two
separate process runs adds to both complexity and expense. Conversely,
when a single microfluidic platform arrange to simultaneously support
both types of genetic testing genotyping and copy number analysis, and
efficiency is realized and cost savings are made possible.

[0052] In accordance with each of preferred embodiments of the invention,
dual mode genetics testing apparatus and methods are provided. It will be
appreciated that each of the embodiments described include an apparatus
and that the apparatus of one preferred embodiment may be different than
the apparatus of another embodiment. Accordingly, limitations read in one
example should not be carried forward and implicitly assumed to be part
of an alternative example.

[0053] One will now fully appreciate how single platform microfluidic
arrays may be configured and deployed to accomplish copy number variation
and genotyping genetic testing in single process runs. Although the
present invention has been described in considerable detail with clear
and concise language and with reference to certain preferred versions
thereof including best modes anticipated by the inventors, other versions
are nevertheless possible. Therefore, the spirit and scope of the
invention should not be limited by any of the preceding descriptions of
preferred versions, but rather by the claims appended hereto.